Mutations in LRRK2 have been linked to both a familial (PARK8) and apparently sporadic forms of PD. At least nine dominantly inherited mutations in LRRK2 have been linked to a late-onset PD to date. The LRRK2 protein, also known as Dardarin, contains multiple functional domains, including a leucine-rich repeat (LRR) domain, a GTPase domain, a kinase domain, and a WD40 domain, may likely function as both an active GTPase and kinase. The most common mutation in LRRK2 is the G2019S substitution at the conserved Mg++-binding motif within the kinase domain, which likely increases the kinase activity of LRRK2. Furthermore, mutant forms of LRRK2 are toxic when over-expressed in cultured cells, and the toxicity likely depends on LRRK2 having kinase activity. The frequency of LRRK2-linked PD has resulted in the availability of post-mortem examinations of brain tissues from disease-affected individuals, unlike DJ-1 and PINK1 inherited mutations. Four autopsy reports stemming from a LRRK2 R441C mutation show evidence of nigral cell loss, and two show LB pathology, while the others present tau-positive neurofibrillary tangles and senile plaques. Several G2019S cases have been autopsied as well, and show typical LB pathology in the brainstem and occasional senile plaques more usually/commonly associated with Alzhemiers disease. Mutations in the kinase domain appear to augment kinase activity and lead to aggregated protein bodies in vitro, suggesting a probable gain-of-function mechanism. There is also evidence that the GTPase domain has a role regulating the kinase activity of the protein, but it is unclear why some of the disease-linked mutations appear to have no effect on either kinase or GTPase activity. Understanding the links between the heterogeneity in pathology from LRRK2 cases and the different domain functions of the protein may help in elucidating the etiology of this more commonly identified form of PD. To better understand the function of LRRK2/Dardarin and pathogenic mechanisms of mutant LRRK2, our specific aims for this project are: Aim 1: To develop and characterize LRRK2 conditional knockout mice Aim 2: To develop and characterize wild-type (WT) and G2109S mutant LRRK2 conditional transgenic mice Aim 3: To investigate the physiological function of LRRK2 and the pathogenic mechanism of mutant LRRK2 We have successfully generated LRRK2 conditional KO mice by flanking the second coding exon with two LoxP sites. Removal of this exon by Cre recombinase causes a frame-shift of LRRK2 coding sequence and generates a premature stop codon in exon 3. Two ES clones carrying the correct homologous recombination of the targeting vector were isolated that gave rise to 6 chimeric mice. All chimeric mice gave birth to F1 heterozygous LRRK2 conditional KO mice, which were then crossed with EIIa-Cre transgenic mice to generate LRRK2 heterozygous KO mice. LRRK2 homozygous KO mice were obtained via intercrossing heterozygous KO mice. The loss of LRRK2 in homozygous KO mice were confirmed by Western blot in which LRRK2 was undetectable in homozygous KO and significantly decreased in heterozygous KO samples. Offspring from the intercross of LRRK2 heterozygous KO mice were born according to the Mendelian rate. LRRK2 homozygous KO mice are viable, developed normally, and have not yet displayed any noticeable behavioral abnormalities up to 12 months of age. There was no apparent difference in body weight between genotypes. The spontaneous ambulatory activity in three age groups (3, 6 and 9 months) of mice was examined by a motion sensitive Flex-Field activity system. ANOVA test indicated that no significant difference existed between genotypes. Similarly, there was also no significant deference in Rotarod tests between genotypes. To examine if there were any neuropathological abnormalities in LRRK2 homozygous KO mice, we checked the gross anatomy of brains, particularly, the morphology anddensity of nigrostriatal dopaminergic (DA) neurons in the midbrain and medium size spiny neurons (MSN) in the striatum. No apparent alteration of cell morphology was observed in LRRK2 homozygous KO mice at 12 months of age. Additionally, no apparent activation of astrocytes and microglia was observed in the brain of these mice. At biochemical level, we examined the expression of ubiquitinated proteins and alpha-synuclein in LRRK2 KO mice and no significant alteration was found. In the future, we will examine the expression of other PD-related proteins in LRRK2 KO mice. We will also investigate the profile of gene expression in LRRK2 KO mice using microarray analysis, which may help us to identify molecular pathways in which LRRK2 is involved. To develop LRRK2 WT and G2019S conditional transgenic mice, a cDNA fragment encoding the C-terminal Hemagglutinin (HA)-tagged human WT or G2019S mutant LRRK2 protein was inserted into pPrP-tetP gene expression vector in which the gene expression is controlled by the tetracycline-responsive promoter (tetP). The LRRK2 expression construct was then purified and microinjected into fertilized oocytes derived from C57BL6/J mice. The founder mice were crossed with NTg C57BL6/J mice to produce the F1 generation. The F1 LRRK2 WT and G2019S mutant mice were mated with CamKII-tTA mice to achieve high expression of LRRK2 in forebrain regions, including the olfactory bulb, striatum, hippocampus, and cortex. We have obtained multiple lines of LRRK2 WT and G2019S transgenic mice. The expression pattern of LRRK2 under the CamKII promoter was confirmed by in situ hybridization using a 33P-labeled oligo probe specific to human LRRK2. As expected, the LRRK2 transgene was selectively expressed in neurons located in the forebrain regions. In addition, a small fraction (<10%) of LRRK2 trangene was expressed in midbrain DA neurons. The expression level of LRRK2 protein was quantified by Western blot using a LRRK2 specific antibody. Both of LRRK2 WT and G2019S transgenic mice were viable, developed normally, and have not yet displayed any noticeable behavioral abnormalities up to 12 months of age. No apparent alteration of neuron morphology was observed in these mice at 12 months of age, either. Additionally, no apparent activation of astrocytes and microglia was observed in the brain of these mice. At biochemical level, we examined the phosphorylation of alpha-synuclein and tau in these mice and no significant alteration was found in these animals as compared with littermate controls. We will keep examining the potential behavioral and neuropathological abnormalities in aged animals. In particular, we will examine the structure and morphology of striatal neurons in LRRK2 KO mice because LRRK2 is highly expressed by these cells and may be involved in the development of nigrostriatal circuitry. Cell biology studies indicate that LRRK2 may regulate the outgrowth of dendrites and axons during neuron morphogenesis. Both RNAi knock-down and genetic knockout of LRRK2 lead to an increase of neurite outgrowth. However, the underlying molecular mechanism is unclear. We will continue to pursue this line of research and to identify the intracellular signaling pathway in which LRRK2 is involved. We have demonstrated a correlation between the dose of LRRK2 and the phosphorylation states of ERM protein in neurons. We will next investigate whether the modification of ERM proteins plays a major role in regulating the outgrowth of neurites in LRRK2-deficient or over-expressing neurons. In addition, we will expand our research on other molecular factors affected by LRRK2 expression during neuron morphogenesis. For example, we will examine the change of gene expression in the brains of newborn LRRK2 WT and G2019S transgenic, and KO pups to identify genetic pathways altered in the mutant LRRK2 mice.